2-O-(β-D-glucopyranosyl) ascorbic acid, process for its production, and foods cosmetics containing compositions comprising it

ABSTRACT

The present invention provides a novel ascorbic acid derivative as a provitamin C with improved stability in the body and prolonged life in the body compared to conventionally known 2-O-(β-D-glucopyranosyl)ascorbic acid. The composition comprising the novel compound 2-O- (β-D-glucopyranosyl)ascorbic acid has been extracted from plants such as from Ningxia  Lycium barbarum  L. and/or  Lycium chinense  Mill. The compositions comprising 2-O-(βD-glucopyranosyl) ascorbic acid may be enzymatically synthesized using β-D-glucosyltransferase. Pure 2-O-(β-D-glucopyranosyl)ascorbic acid may be produced from such compositions. Alternatively, 2-O-(β-D-glucopyranosyl)ascorbic acid may be produced by chemical synthesis. The 2-O-(β-D-glucopyranosyl)ascorbic acid results in higher stability and a prolonged life of vitamin C when ingested in the body compared to the corresponding α-D-glucopyranosyl derivative, and is therefore highly suitable as a provitamin C to be used in cosmetics and foods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/500,334, filed Dec. 30, 2004, now U.S. Pat. No. 7,566,698, which is anational stage of International Application No. PCT/JP02/13857 filedDec. 27, 2002, and which claims benefit of Japanese Patent ApplicationNo. 2001-400258 filed on Dec. 28, 2001; the entire contents of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a novel provitaminC,2-O-(β-D-glucopyranosyl)ascorbic acid, which has an increasedstability, an extended half-life and a long lasting activity in the bodycompared to known provitamin C substances such as2-O-(α-D-glucopyranosyl)ascorbic acid. The invention further relates toa novel intermediate, 2-O-(tetra-O-acetyl-β-D-glucopyranosyl) ascorbicacid, to a production process for 2-O-(β-D-glucopyranosyl)ascorbic acidusing the 2-O-(tetra-O-acetyl-β-D-glucopyranosyl)ascorbic acidderivative as an intermediate, to a production process for2-O-(β-D-glucopyranosyl)ascorbic acid extracted from a plant, andparticularly a production process for 2-O-(β-D-glucopyranosyl)ascorbicacid extracted from a plant of Lycium genus, L. chinense Mill., L.barbarum L. or its related species, to compositions comprising theobtained 2-O-(β-D-glucopyranosyl)ascorbic acid, to an enzymaticproduction process for 2-O-(β-D-glucopyranosyl)ascorbic acid orcompositions comprising 6-O-(β-D-glucopyranosyl)ascorbic acid and2-O-(β-D-glucopyranosyl)ascorbic acid, and to the use of suchcompositions as foods or cosmetics.

PRIOR ART

Vitamin C is known to have numerous physiological effects, such ascollagen synthesis, the lack of which is the major causative factor ofscurvy, acting as a biological antioxidant to eliminate free radicalsproduced in the body and contributing to the iron ionoxidation-reduction reaction of cytochrome C, as well as havinganticancer, immunoactivating, and cholesterol production-inhibiting andtherefore anti-arteriosclerotic effects. For skin, it exhibits variouseffects including photoaging inhibition, ultraviolet damage preventionand pigmentation inhibition, through antioxidation and collagensynthesis promotion, and is therefore used as an additive in cosmeticproducts (Fragrance Journal, Vol. 25, Special Issue, Mar. 1997). It isalso added to foods and cosmetics as an antioxidant. One of the majordrawbacks of vitamin C is its extreme instability with respect to light,heat, oxygen and metal ions.

Various modifications have been studied to improve the instability ofvitamin C or alter its properties to improve its retention andabsorption in the body (Nihon Rinsho, Vol. 57, No. 10, p. 170, 1999) Ithas been attempted to introduce substituents at the hydroxyl groups ofthe 2,3-enediol, which is the antioxidizing moiety of vitamin C and thesource of its instability, toward the aim of developing a more stableform of vitamin C known as “provitamin C”. Examples include introductionof sulfate groups (Biochemistry, 8, 2652, 1969) or phosphate groups(Gazz. Chim. Ital., 91, 964, 1961 and Chem. Pharm. Bull., 17, 381, 1969)at the 2-hydroxyl position. Such compounds have greatly enhancedstability compared to ordinary vitamin C, and are known to be convertedto vitamin C by in vivo and intracellular hydrolysis by sulfatases orphosphatases. These derivatives are already used in cosmetics and quasidrugs. Stable forms of vitamin C are also known which are glycosylatedat the 2- or 3-hydroxyl groups. These include2-O-(α-D-glucopyranosyl)ascorbic acid obtained with glycosyltransferase(Biochim. Biophys. Acta, 1035, 44, 1990, Japanese Unexamined PatentPublication HEI Nos. 3-135992, 3-139288, 3-183492, 5-117290),2-O-(β-D-galactopyranosyl)ascorbic acid obtained with galactosidase(Japanese Unexamined Patent Publication HEI No. 6-263790) and3-O-(β-D-glucopyranosyl)ascorbic acid obtained by chemical synthesis(Japanese Unexamined Patent Publication SHO Nos. 53-98954, 58-198498),and the like.

Among these, most research has been conducted on2-O-(α-D-glucopyranosyl)ascorbic acid, which is currently used incosmetics and quasi drugs and under examination for approval as a foodadditive. 2-O-(α-D-Glucopyranosyl)ascorbic acid is, similar toascorbyl-2-phosphate, highly stable under various oxidative conditions,and is much more stable under acidic conditions. When taken orally,2-O-(α-D-glucopyranosyl)ascorbic acid is hydrolyzed by α-glucosidasepresent in the gastrointestinal mucosa and converted to the active formof vitamin C. It is also moderately hydrolyzed by enzymes present in thecell membranes of cultured cells whereby the action of vitamin C isexhibited continuously.

While unrelated to improvement in stability against oxidation, fattyacid esters of ascorbic acid at the 6-position, such as 6-O-palmitoyland stearoylascorbic acids which are readily soluble in fat-solublesubstances, are used as food antioxidants in food additives. Also,6-glycosides have been synthesized enzymatically (Vitamins, 43, 205,1971; Biochim. Biophys. Acta, 1035, 44, 1990;, Japanese UnexaminedPatent Publication HEI No. 5-320185). 5-Glycosides have also beendisclosed, as by-products of 2-O-(α-D-glucopyranosyl)ascorbic acid byenzymatic synthesis (Japanese Unexamined Patent Publication HEI No.5-112594).

Thus, numerous provitamin C substances are already known, butβ-D-glucosides, particularly 2-O-glucoside substances, especially thenovel 2-O-(β-D-glucopyranosyl)ascorbic acid compound of the presentinvention, have been unknown. And documents such as Japanese UnexaminedPatent Publication HEI No. 3-13599, which discloses thatβ-D-glucopyranosyl L-ascorbic acid derivatives cannot be decomposed inthe body, teach that β-glucosides cannot be utilized in the body and aretherefore not useful.

Japanese Unexamined Patent Application No. Sho 53-98954 describes2-O-(β-D-glucopyranosyl)ascorbic acid along with various ascorbic acidderivatives, but provides no concrete production examples, and it isbelieved that even with actual synthesis by the process in thoseexamples, the 3-hydroxyl groups would be preferentially glucosylated,subsequently producing 2,3-diglucosides with glucosylation at the2-position with respect to the 3-glucosylated product. It shouldtherefore be impossible to obtain a product with β-glucosylation only atthe 2-position.

The only report of enzymatic synthesis of β-D-glucopyranosyl L-ascorbicacid derivatives is that of production of6-O-(β-D-glucopyranosyl)ascorbic acid by β-glucosidase from almond usingcellobiose as the β-glucosyl donor (Agric. Biol. Chem., 54, 1697, 1990).In this case, the transfer yield of 6-O-(β-D-glucopyranosyl)ascorbicacid is a very low value of 1.5%, and there is no mention of productionof 2-O-(β-D-glucopyranosyl)ascorbic acid. Thus, absolutely no case hasbeen known where 2-O-(β-D-glucopyranosyl)ascorbic acid is synthesized byan enzymatic method.

Lycium chinense Mill. (Chinese matrimony-vine), a plant of theSolanaceae family, is listed as a delicacy in the ancient Chinesemedical text “Compendium of Materia Medica”, and the fruit thereof,known as lycii fructus and the leaves known as lycii folium are used asfoods while the root skin, known as lycii cortex radicis, is used as aChinese herbal medicine (Genshoku Wakanyaku Zukan [IllustratedCompendium of Oriental Drugs], Vol. I, 289, 1980). Lycium chinense Mill.contains betaine, carotene, nicotinic acid and zeaxanthin, and is knownto have hypoglycemic, anti-hypertensive, lipotropic and hepaticfunction-protecting effects. In particular, the lipotropic and hepaticfunction-protecting effects are attributed to betaine, which acts as amethyl group donor (Folia Pharmacol. Japon, 56, 151, 1960). Also, aplant of Lycium genus extract is known to promote the growth and acidproduction of lactic acid bacteria (C.A. 64:20530b, 1965). However, thepresence of vitamin C derivatives among the components in a plant ofLycium genus has been unknown.

SUMMARY OF THE INVENTION

As a result of extensive research focused on the myriad effects of aplant of Lycium genus and its active components, the present inventorsdiscovered a new substance contained therein, and completed one aspectof the invention by determining that the substance is2-O-(β-D-glucopyranosyl)ascorbic acid. The invention therefore providesthe novel substance-2-O-(β-D-glucopyranosyl)ascorbic acid, a compositioncomprising 2-O-(β-D-glucopyranosyl)ascorbic acid extracted from a plantof Lycium genus and a process for their production.

The novel substance of the invention is useful as a provitamin C.β-Glucosidase is known to be present in membrane-bound form in smallintestine tissue and in cytoplasmic form in hepatic and renal tissue(FEBS Letters, 436, 71, 1998), whereas α-glucosidase is widelydistributed in saliva, intestinal digestive juices and the smallintestinal tract where it presumably decomposes substrates to ascorbicacid upon peroral uptake. A report by Yamamoto et al. (J.Pharmacobio-Dyn. 13, 688, 1990) describes that only ascorbic acid isdetected in the blood in an experiment with oral administration to rats,thus suggesting that instead of activation by α-glucosidase which iswidely distributed in the body, decomposition and activation byβ-glucosidase, which is less widely distributed, would be moreadvantageous in terms of the transport into tissues and long-lastingaction; β-glucosides of provitamin C are therefore expected to exhibiteven more desirable properties.

Upon studying the activity of the novel compound of the invention,2-O-(β-D-glucopyranosyl) ascorbic acid, it was found to be highly usefulas a provitamin C due to the improved stability and the prolonged effectin the body compared to 2-O-(α-D-glucopyranosyl)ascorbic acid. Inaddition, processes for its industrial production for utilization infoods and cosmetics were studied, and the invention was completed uponestablishing a production process by chemical synthesis and extractionfrom natural plants, as well as an enzymatic production process.

Accordingly, the invention provides the novel substance2-O-(β-D-glucopyranosyl)ascorbic acid, which has physiological actionsuperior to that of 2-O-(α-D-glucopyranosyl)ascorbic acid and isexpected to have applications in the fields of cosmetics, quasi drugs,medicines and foods, as well as2-O-(tetra-O-acetyl-β-D-glucopyranosyl)ascorbic acid as a novelintermediate thereof, a process for production of2-O-(β-D-glucopyranosyl)ascorbic acid by chemical synthesis through theintermediate, a process for production of compositions comprising2-O-(β-D-glucopyranosyl)ascorbic acid by extraction from plants andespecially from a plant of Lycium genus, L. chinense Mill., L. barbarumL., a process for production of compositions comprising2-O-(β-D-glucopyranosyl)ascorbic acid using β-D-glucosyltransferase,compositions comprising the obtained 2-O-(β-D-glucopyranosyl)ascorbicacid, and foods or cosmetics containing the compositions.

The invention further provides compositions comprising2-O-(β-D-glucopyranosyl)ascorbic acid or6-O-(β-D-glucopyranosyl)ascorbic acid obtained by the reaction usingglycosyltransferase. It still further provides a method for easy removalof contaminants from solutions containing2-O-(β-D-glucopyranosyl)ascorbic acid and an industrial scale productionprocess of products with higher contents and higher purity of2-O-(β-D-glucopyranosyl)ascorbic acid.

Throughout the present specification, the term “composition” will referto any of various compositions comprising2-O-(β-D-glucopyranosyl)ascorbic acid, including extracts with increased2-O-(β-D-glucopyranosyl)ascorbic acid contents from plants containing2-O-(β-D-glucopyranosyl)ascorbic acid, or reaction products containing2-O-(β-D-glucopyranosyl)ascorbic acid which are obtained by reactingascorbic acid and β-D-glucoside compounds using β-glucosyltransferase.

Throughout the present specification, the term “provitamin C” willcollectively refer to compounds which themselves exhibit weak or novitamin C activity but are decomposed in the body to produce vitamin C,as well as compositions comprising such compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of ion-exchange chromatography of2-O-(β-D-glucopyranosyl)ascorbic acid extracted from lycii fructus.

FIG. 2 shows the results of further purifying2-O-(β-D-glucopyranosyl)ascorbic acid by high performance liquidchromatography using a portion of fractions 19-25 of FIG. 1, andcomparing the ¹H-NMR with a chemically synthesized product. The upperchart is the spectrum for lycii fructus-derived2-O-(β-D-glucopyranosyl)ascorbic acid, and the lower chart is thespectrum for chemically synthesized 2-O-(β-D-glucopyranosyl) ascorbicacid.

FIG. 3 shows the results of comparing enzymatically synthesized2-O-(β-D-glucopyranosyl)ascorbic acid (substance Y) with a chemicallysynthesized product based on ¹H-NMR. The upper chart is the spectrum forchemically synthesized 2-O-(β-D-glucopyranosyl)ascorbic acid, and thelower chart is the spectrum for the enzymatic reaction product2-O-(β-D-glucopyranosyl)ascorbic acid (substance Y).

FIG. 4 shows the HSQC spectrum for enzymatically synthesized6-O-(β-D-glucopyranosyl)ascorbic acid (substance X), in comparison withchemically synthesized 2-O-(β-D-glucopyranosyl)ascorbic acid. FIG 4A isthe spectrum for the enzyme reaction product6-O-(β-D-glucopyranosyl)ascorbic acid (substance X), and FIG. 4B is thespectrum for chemically synthesized 2-O-(β-D-glucopyranosyl)ascorbicacid.

FIG. 5 shows the protective effect of previous administration with2-O-(β-D-glucopyranosyl)ascorbic acid against cell death of human skinepidermal keratinocytes (HaCaT) induced by ultraviolet B (UVB)irradiation.

FIG. 6 shows the effect of 2-O-(β-D-glucopyranosyl) ascorbic acid onintracellular ascorbic acid concentration in human skin epidermalkeratinocytes.

FIG. 7 shows the promoting effect of 2-O-(β-D-glucopyranosyl)ascorbicacid on collagen synthesis by normal human dermal fibroblasts (NHDF).

FIG. 8 shows the rat portal blood and plasma concentrations after oraladministration of 2-O-(β-D-glucopyranosyl)ascorbic acid at 100 mg/kg.

FIG. 9 shows the effect of 2-O-(β-D-glucopyranosyl)ascorbic acid onpopulation doubling levels (PDLs) of normal human dermal fibroblast(NHDF) cells. 2-O-(β-D-Glucopyranosyl)ascorbic acid showed a remarkablelife-supporting effect. Namely, while NHDF died of aging at PDL of 20.1in the absence of ascorbic acid derivative, the PDL of NHDF was greaterby 2.62 times in the presence of said ascorbic acid derivative. Theresults indicate that the lifelong cell-supplying ability of NHDF wasmade greater by 2^(2.62), and the ascorbic acid derivative of theinvention will make a great contribution in protecting the skin fromaging and compensating dead cells in the skin.

FIG. 10 shows the effect of 2-O-(β-D-glucopyranosyl)ascorbic acid on thereduction speed of telomere lengths. The average reduction speed 225bp/PDL in the absence of any ascorbic acid derivative was shortened to94 bp/PDL by the addition of 2-O-(β-D-glucopyranosyl)ascorbic acid.Since the critical telomere length at the time of cell death by aging isknown to be constant, it is considered that the ascorbic acid derivativeof the invention exhibited a prolonging effect on the life span of thecell line by shortening the speed, by approximately 42%, of the telomerelength to reduce to the critical length.

DESCRIPTION

The present inventors conducted extensive research on methods ofsynthesizing 2-O-(β-D-glucopyranosyl)ascorbic acid with the aim ofcreating the ideal provitamin C with the novel substance2-O-(β-D-glucopyranosyl)ascorbic acid discovered in a plant of Lyciumgenus, and found that chemical production is possible using2-O-(tetra-O-acetyl-β-D-glucopyranosyl)ascorbic acid as an intermediate.Upon further ardent examination to search for plants and microbescontaining 2-O-(β-D-glucopyranosyl)ascorbic acid, it was found that2-O-(β-D-glucopyranosyl)ascorbic acid is present in a plant of Lyciumgenus and lycii fructus. They also found that2-O-(β-D-glucopyranosyl)ascorbic acid is produced by glycosyltransferreaction of cellulase enzymes, and that 2-O-(β-D-glucopyranosyl)ascorbicacid is in fact an excellent provitamin C which exhibits notableinhibition of cell death upon ultraviolet B irradiation of human skinepidermal keratinocytes and notable promotion of collagen synthesis innormal human dermal fibroblasts, as compared to2-O-(α-D-glucopyranosyl)ascorbic acid. The present invention has beencompleted upon these findings, and is based on the following.

Production of 2-O-(β-D-glucopyranosyl)ascorbic acid, which exhibits moreexcellent performance as a provitamin C than2-O-(α-D-glucopyranosyl)ascorbic acid, can be achieved by a synthesismethod using 2-O-(tetra-O-acyl-β-D-glucopyranosyl)ascorbic acid as anintermediate, by an extraction method from natural materials containing2-O-(β-D-glucopyranosyl)ascorbic acid, and by an enzymatic synthesismethod.

In the synthesis method, the 3-hydroxyl group of the correspondingascorbic acid derivative is selectively benzylated to obtain3-O-benzyl-5,6-O-isopropylideneascorbic acid, which is condensed with anester-protected glucose 1-carbonate ester and then de-isopropylidenatedand de-benzylated to obtain the product in the extraction method from anatural material, a composition comprising2-O-(β-D-glucopyranosyl)ascorbic acid may be obtained by extraction inhot water or water-alcohol from a plant of the family Solanaceae, andparticularly the fresh fruit or dried fruit (lycii fructus) of a plantof Lycium genus. If necessary, the 2-O-(β-D-glucopyranosyl)ascorbic acidmay be further purified from such a composition.2-O-(β-D-glucopyranosyl)ascorbic acid is also enzymatically synthesizedby glycosyltransfer reaction of a cellulase to obtain a compositioncomprising 2-O-(β-D-glucopyranosyl)ascorbic acid. If necessary, the2-O-(β-D-glucopyranosyl) ascorbic acid may be further purified from sucha composition.

As it has been demonstrated by the present invention that2-O-(β-D-glucopyranosyl)ascorbic acid notably inhibits cell death uponultraviolet B irradiation of human skin epidermal keratinocytes andnotably promotes collagen synthesis in normal human dermal fibroblasts,and that it is intracellularly converted to vitamin C and absorbed byoral ingestion, it is therefore expected to be useful as a skin cosmeticor skin protective agent and useful in foods as a provitamin C.

Preferred Embodiment

The invention provides 2-O-(β-D-glucopyranosyl)ascorbic acid, which hasphysiological action superior to 2-O-(α-D-glucopyranosyl)ascorbic acidand is expected to have applications in the fields of cosmetics, quasidrugs, medicines and foods, as well as2-O-(tetra-O-acetyl-β-D-glucopyranosyl)ascorbic acid as an intermediatethereof, a process for production of 2-O-(β-D-glucopyranosyl)ascorbicacid using the intermediate, and 2-O-(β-D-glucopyranosyl)ascorbicacid-containing compositions comprising extracts from a plant of Lyciumgenus, L. chinense Mill., L. barbarum L. or its-related species whichcontain 2-O-(β-D-glucopyranosyl)ascorbic acid.

The invention further provides compositions comprising2-O-(β-D-glucopyranosyl)ascorbic acid or6-O-(β-D-glucopyranosyl)ascorbic acid obtained by glucosyltransferasereaction. It still further provides a method for easy removal ofcontaminants from solutions containing 2-O-(β-D-glucopyranosyl)ascorbicacid and an industrial scale production process of products with highercontents and higher purity of 2-O-(β-D-glucopyranosyl)ascorbic acid.

The invention will now be explained in greater detail.

1) Synthesis of Intermediate:2-O-(2,3,4,6-tetra-O-acyl-β-D-glucopyranosyl)ascorbic acid

The intermediate 2-O-(2,3,4,6-tetra-O-acyl-β-D-glucopyranosyl)ascorbicacid may be synthesized in the following manner. Specifically,commercially available 5,6-O-isopropylideneascorbic acid is selectivelybenzylated at the 3-hydroxyl position by a known method (J. Med. Chem.,31, 793, 1988), to produce 3-O-benzyl-5,6-O-isopropylideneascorbic acid.This 3-O-benzylated compound as the aglycone is glycosylated by ordinaryglycosylation reaction, to obtain2-O-(2,3,4,6-tetra-O-acyl-β-D-glucopyranosyl)-3-O-benzyl-5,6-O-isopropylideneascorbicacid. For example, a (2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)carbonicacid ester (Komura, H., Tokyo Institute of Technology doctoral thesis,1977) may be obtained by heating at 100-200° C. together with the3-O-benzylated compound in a nonpolar solvent or without a solvent. Thecarbonic acid ester used may be an alkyl-, halogenated alkyl- oroptionally substituted aryl-carbonic acid ester. Alternatively, a(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)halide may be used forreaction in a halogenated hydrocarbon solvent such as chloroform ormethylene chloride or an aromatic hydrocarbon solvent such as benzene ortoluene, in the presence of a mercury salt or silver salt with additionof a dehydrating agent (Lodd's Chemistry of Carbon Compounds IF, 320,1967, Elsevier).

The isopropylidene group of2-O-(2,3,4,6-tetra-O-acyl-β-D-glucopyranosyl)-3-O-benzyl-5,6-O-isopropylideneascorbicacid may be hydrolyzed with an acid catalyst for its removal. Forexample, deisopropylidenation may be performed at 40-100° C. in a 30-80%acetic acid aqueous solution. Alternatively, it may be performed fromroom temperature to reflux temperature in acetone or methyl ethyl ketonein the presence of p-toluenesulfonic acid. Water may instead be used forthe same reaction.

The benzyl group of the2-O-(2,3,4,6-tetra-O-acyl-β-D-glucopyranosyl)-3-O-benzylascorbic acidmay be removed by ordinary hydrogenolysis. For example, debenzylationmay be accomplished in a protic polar solvent such as acetic acid oralcohol, or a non-polar solvent such as benzene, toluene or ethylacetate, in the presence of hydrogen, using palladium-carbon, palladiumblack, platinum-carbon or platinum black as the catalyst.

The deprotection steps may be carried out in the reverse order. That is,after debenzylation reaction of the2-O-(2,3,4,6-tetra-O-acyl-β-D-glucopyranosyl)-3-O-benzyl-5,6-O-isopropylideneascorbicacid, the resulting2-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-5,6-O-isopropylideneascorbicacid may be deisopropylidenated with an acid catalyst.

The title intermediate,2-O-(2,3,4,6-tetra-O-acyl-β-D-glucopyranosyl)ascorbic acid, may beobtained in the manner described above.

Acetyl is preferred as the acyl group of the title intermediate.

2) Chemical Synthesis of 2-O-(β-D-glucopyranosyl)ascorbic acid

2-O-(β-D-Glucopyranosyl)ascorbic acid may be obtained by alkalihydrolysis of the acyl group of2-O-(2,3,4,6-tetra-O-acyl-β-D-glucopyranosyl)ascorbic acid. The alkaliused may be an aqueous solution of sodium hydroxide or potassiumhydroxide, an aqueous solution of carbonate such as potassium carbonate,sodium carbonate, potassium bicarbonate or sodium bicarbonate, or ametal alcoholate such as sodium methylate. The solution may contain analcohol such as methanol and ethanol to dissolve the2-O-(2,3,4,6-tetra-O-acyl-β-D-glucopyranosyl)ascorbic acid as thestarting material. The reaction temperature is optimally from 0° C. toroom temperature. The reaction solution is neutralized with hydrochloricacid, sulfuric acid or a cation-exchange resin. In the case ofhydrochloric acid or sulfuric acid it is necessary to remove the saltwhich is produced, but in the case of a cation-exchange resin nodesalting procedure is required due to the adsorption of sodium andpotassium salts. The neutralized solution may be lyophilized orconcentrated under reduced pressure to obtain the desired compound.Depending on the purpose, the compound may be purified further by columnchromatography.

3) Production of Composition Comprising 2-O-(β-D-glucopyranosyl)ascorbicacid by Extraction from a Plant of Lycium genus, L. chinense Mill., L.barbarum L.

The fresh or dried fruit (lycii fructus) of a plant of Lycium genus, L.chinense Mill., L. barbarum L. is immersed in an aqueous solvent such ashot water or aqueous ethanol either directly or after pulverization, andthe extract obtained by solid/liquid separation is concentrated underreduced pressure or lyophilized, or else spray dried, to obtain anextract containing 2-O-(β-D-glucopyranosyl)ascorbic acid. The alcoholconcentration during the immersion is preferably from 10-95%, and theimmersion is preferably continued from 3 to 7 days.

The 2-O-(β-D-glucopyranosyl)ascorbic acid content in the lycii fructusextract will typically be from 0.86 to 1.2%, but a composition with aneven higher content can be obtained by the method described hereunder.Specifically, the lycii fructus extract is dissolved in distilled water,or the extract obtained by immersing the starting material in 5-50volumes and preferably 8-10 volumes of the solvent is diluted withdistilled water, and then passed through a strongly basic anion exchangeresin such as Dowex™ 1-X8 (Dow Chemical Co.) or Amberlite IRA-400 (Rohm& Haas Co.) for adsorption of the 2-O-(β-D-glucopyranosyl)ascorbic acid.After thorough washing with water, a fraction containing the desiredsubstance is obtained by stepwise elution or gradient elution using anacid solution of acetic acid or the like. The fraction is concentratedunder reduced pressure or lyophilized to remove acetic acid, thereby toyield a composition containing 2-O-(β-D-glucopyranosyl)ascorbic acid atapproximately 30-50%.

4) Production of Composition Comprising 2-O-(β-D-glucopyranosyl)ascorbicacid by Enzyme Method

As a result of extensive study on commercially available enzymepreparations, it was found that β-glucosyltransferase activity isexhibited by the enzyme preparations cellulase “Onozuka” and PancelaseBR (Yakult Pharmaceutical Ind. Co., Ltd.), Cellulosin (Hankyu KyoeiBussan), Cellulase (Sigma), β-glucosidase (Toyobo) and β-glucosidase(Nacalai Tesque). The glycosyltransferase used for the invention may beany one which acts on solutions comprising β-glucosyl group-containingcompounds and ascorbic acid to synthesize2-O-(β-D-glucopyranosyl)ascorbic acid by glycosylation reaction, and thesource and type is not limited; however, from the standpoint of yield,cellulases from Trichoderma and β-glucosidases from almond arepreferred.

The cellobiose and ascorbic acid concentrations for the transferasereaction are preferably as high as possible, and preferably about 0.3 Mand 0.2 M, respectively. The cellobiose as the enzyme substrate may besupplied as another β-glucosyl group-containing compound, such as a highmolecular glucan such as cellulose or carboxymethyl cellulose incombination with an appropriate hydrolase. Each enzyme may beimmobilized on a suitable support by an ordinary method to form anenzyme reactor to facilitate efficient production of2-O-(β-D-glucopyranosyl)ascorbic acid. On the other hand, the ascorbicacid serving as the acceptor in the transfer reaction is preferably thefree acid from the standpoint of stability and transfer yield in thereaction, but ascorbic acid may be used also in the form of a salt suchas an alkali metal salt or an alkaline earth metal salt, or a mixturethereof. It was further discovered that isoascorbic acid, either thefree acid form or a salt thereof, likewise act as the acceptor in thetransfer reaction. Consequently, ascorbic acid or ascorbic acidderivatives may, be used for the transglycosylation reaction dependingon the purpose, and in most cases sodium ascorbate, calcium ascorbateand the like may be suitably used according to the need, instead of freeascorbic acid alone.

The enzyme reaction proceeds in aqueous solution in a pH range of 2-8,but preferably at pH 4-6 considering the optimum pH for the enzyme. Thereaction temperature will be 20-60° C., but is preferably kept at about30-40° C. considering the enzyme stability and optimum temperature. Theamount of enzyme added is preferably 20-400 units (where 1 unit is theactivity of the enzyme which liberates 1 μmol of p-nitrophenol perminute) per gram of cellobiose. The enzyme may be added all at once, butit may also be added several times while monitoring the reaction by highperformance liquid chromatography. The reaction may also be conducted byimmobilizing the enzyme on an appropriate resin substrate such as anion-exchange resin or hydrophobic resin as an enzyme reactor. Thereaction time of about 1-4 days will be sufficient, but completion pointof the reaction may be determined while monitoring the reaction.

The ascorbic acid derivative in the composition produced at thecompletion of the reaction may be further purified if desired byordinary separating means such as membrane separation, ion-exchangecolumn chromatography, activated carbon column chromatography, liquidchromatography, silica gel column chromatography or the like. Forexample, as a strongly acidic cation-exchange resin, there may beappropriately used an alkali metal salt-type, alkaline earth metalsalt-type or H⁺-type of a sulfonated styrene-divinylbenzene crosslinkedcopolymer resin. Commercially available products include Dowex™ 50W×8(the Dow Chemical Company), Amberlite™ CG-120 (Rohm & Haas Co.) andDiaion™ SK104 (Mitsubishi Chemical Industries Co., Ltd.). The unreactedascorbic acid and β-glucosyl group-containing compound separated by thechromatography may be used as starting materials in the next round ofenzyme reaction.

In order to achieve higher product purity, purification may be performedby high performance liquid chromatography. Specifically, a pure productmay be obtained by employing a combination of a sugar/organic acidanalyzing column and a volatile acid such as acetic acid,trifluoroacetic acid, and the like, or a combination of an ODS columnwith sublimating ammonium formate, and a volatile ion-pair reagentdi-n-butylamine acetate which is used to analyze acidic substances.Identification of the target substance may be accomplished bycomparative analysis of the mass spectrum or nuclear magnetic resonancespectrum against chemically synthesized 2-O-(β-D-glucopyranosyl)ascorbicacid.

The 2-O-(β-D-glucopyranosyl)ascorbic acid may be used in the form of asalt suitable for foods, cosmetics or drugs. As examples of salts theremay be mentioned inorganic or organic alkali salts, including sodiumsalts, potassium salts, calcium salts and amine salts. The2-O-(β-D-glucopyranosyl)ascorbic acid may be substituted at a hydroxylgroup with a leaving group that is easily degraded in the body. As suchleaving groups there may be mentioned acetyl (C₂), propionyl (C₃),butyryl (C₄), octanoyl (C₈), palmitoyl (C₁₆) and stearoyl (C₁₈).

5) Activity of 2-O-(β-D-glucopyranosyl)ascorbic acid

(1): Inhibition of Ultraviolet Irradiation Damage

2-O-(β-D-glucopyranosyl)ascorbic acid clearly exhibits a stronger effectof preventing cell death of human skin epidermal keratinocytes (HaCaT)induced by ultraviolet B (UVB) irradiation compared to ascorbic acid or2-O-(α-D-glucopyranosyl)ascorbic acid at the same concentration.

It is known that with irradiation on a portion of hairless mouse skinwith light near the wavelength spectrum of sunlight (290-400 nm),reduction of ascorbic acid, (vitamin C) occurs most rapidly among theantioxidant factors in mouse skin (Photodermatol. Photoimmunol.Photomed., 10(5), 183, 1994). Also, skin inflammation induced by UVBirradiation of shaven guinea pig dorsal skin can be inhibited byexternal application of ascorbic acid or2-O-(α-D-glucopyranosyl)ascorbic acid, with the effect of2-O-(α-D-glucopyranosyl)ascorbic acid being greater (Fragrance Journal,Vol. 25, No. 3, p. 55, 1997). Daily administration of 10% ascorbic acidaqueous solution to pig skin for a period from 3 days to a week has alsobeen reported to alleviate ultraviolet ray damage (Br. J. Dermatol.,121, 247, 1992).

However, the effect of the 2-O-(β-D-glucopyranosyl) ascorbic acid of theinvention against the skin inflammation which is induced by ultravioletirradiation and other ultraviolet ray damage is greater than that ofascorbic acid or 2-O-(α-D-glucopyranosyl) ascorbic acid. As mentionedabove, the reason for this is believed to be its superior migration intotissue and longer life compared to 2-O-(α-D-glucopyranosyl)ascorbicacid.

In respect of the intracellular ascorbic acid concentration in humanskin epidermal keratinocytes, 2-O-(β-D-glucopyranosyl)ascorbic acid isalso maintained at a higher concentration for the longest time. Thishigh concentration maintenance of intracellular ascorbic acid by2-O-(β-D-glucopyranosyl)ascorbic acid is attributed to its activity ofprotecting cells against UVB irradiation. It is also clear that2-O-(β-D-glucopyranosyl)ascorbic acid functions as a provitamin C whichis intracellularly converted to ascorbic acid.

Activity of 2-O-(β-D-glucopyranosyl)ascorbic acid

(2): Prevention of Wrinkles and Sagging

Examination of collagen synthesis by normal human dermal fibroblasts(NHDF) indicates increased activity of 2-O-(β-D-glucopyranosyl)ascorbicacid compared to 2-O-(α-D-glucopyranosyl)ascorbic acid or ascorbic acid.This is also believed to be due to the prolonged higher concentration ofintracellular ascorbic acid. That is, the collagen synthesis-promotingeffect of ascorbic acid is believed to occur even in skin-derivedfibroblasts, functioning for regeneration and reconstruction of theskin. In fact, application of ascorbic 2-phosphate, as a stable form ofascorbic acid, to burn victims has been reported to promote healingwithout cicatrization (Lecture Summaries of the Japanese CosmeticScience Society, p. 50, 1998). On the other hand, ascorbic acid is alsoknown to inhibit collagen-degrading enzymes, and enzymes that degradeelastin which is essential for skin elasticity (BioantioxidantProvitamin C, p. 63, 1999, Fragrance Journal Co.). These data suggest aneffect of 2-O-(β-D-glucopyranosyl)ascorbic acid against wrinkles andsagging.

Activity of 2-O-(β-D-glucopyranosyl)ascorbic acid

(3): Whitening

Based on the fact that ascorbic acid inhibits tyrosinases and therebysuppresses melanin synthesis and that human application of creamcontaining 2-O-(α-D-glucopyranosyl)ascorbic acid inhibits pigmentationcaused by ultraviolet irradiation (Fragrance Journal, Vol. 25, No. 3, p.55, 1997), it is strongly suggested that2-O-(β-D-glucopyranosyl)ascorbic acid will have a similar whiteningeffect, and stronger than that of 2-O-(α-D-glucopyranosyl)ascorbic acid.

Activity of 2-O-(β-D-glucopyranosyl)ascorbic acid

(4): Dynamics with oral ingestion

Upon oral ingestion of 2-O-(β-D-glucopyranosyl)ascorbic acid by rats,unconverted 2-O-(β-D-glucopyranosyl)ascorbic acid is detected in theblood, indicating that it is absorbed in unconverted form through theintestinal tract. On the other hand, as mentioned above, oral ingestionof 2-O-(α-D-glucopyranosyl)ascorbic acid by rats results in no detectionof the unconverted form in the blood, and therefore upon absorption itis almost completely decomposed in the intestinal tract and exists inthe blood as ascorbic acid (J. Pharmacobio-Dyn., 13, 688, 1990). Thatis, upon oral ingestion, 2-O-(α-D-glucopyranosyl)ascorbic acid isabsorbed as ascorbic acid and is more likely to be rapidly degraded inthe blood. On the other hand, 2-O-(β-D-glucopyranosyl)ascorbic acidexists in the blood even in its unconverted form and migratesunconverted into the tissue, so that it is more likely to be activatedto ascorbic acid in the tissues and cells.

The above experimental results and observations clearly indicate that2-O-(β-D-glucopyranosyl)ascorbic acid and compositions comprising it arean excellent form of provitamin C useful for protecting skin andmaintaining healthy skin, and may be used in skin cosmetics and skinprotectors or in foods, as a provitamin C permitting efficient migrationof ascorbic acid into the body and tissues.

6) Composition Comprising 2-O-((3-D-glucopyranosyl)ascorbic acid

When a composition comprising 2-O-(β-D-glucopyranosyl)ascorbic acidaccording to the invention is used as a skin cosmetic or skin protector,the amount thereof may be within a wide range with no particularrestrictions, but will ordinarily be 0.1-30 wt % and preferably 0.5-10wt % with respect to the total amount of the composition. In the form ofa composition, it may be appropriately combined with other componentscommonly used in cosmetics, such as oil components, surfactants,ultraviolet absorbers, lower alcohols, preservatives, bacteriocidalagents, coloring agents, powders, aromas, water-soluble polymers,buffering agents and the like, so long as the effect of the invention isnot impaired. Such compositions may be used not only as skin cosmeticsbut also as quasi drugs in the form of lotions, emulsions, creams,packs, soaps or other medicinal cosmetics, or as drugs in the form oflotions, emulsions, creams, ointments or other external skinapplications.

When a composition comprising 2-O-(β-D-glucopyranosyl)ascorbic acid ofthe invention is used as a food, it may be applied in food products inthe same manner as described in Japanese Patent No. 2832848 in regard to2-O-(α-D-glucopyranosyl)ascorbic acid, to prepare vitamin C-fortifiedfoods. To cite Japanese Patent No. 2832848 specifically, because of itscompatibility with various substances of various flavors includingacidic, salty, astringent, zesty, bitter, etc., and its high acidresistance and heat resistance, it may be utilized as a vitaminC-fortifier, flavor enhancer, acidic flavoring, quality improver,stabilizer, antioxidant or the like in various common food products andflavorings, for example, in various types of seasonings such as soysauce, powdered soy sauce, salted bean paste, powdered salted beanpaste, unrefined sake, salted meat, fish flour, mayonnaise, dressing,vinegar, sake/soy/vinegar sauce, powdered sushi vinegar, chineseseasoning, tempura broth, noodle broth, sauce, ketchup, roast meatgravy, curry powder, stew seasoning, soup seasoning, stock seasoning,mixed condiment, sweet sake, low-alcohol sweet sake, table sugar andcoffee sugar; Japanese sweets such as rice crackers, rice-cake cubes,millet/rice cake, fried dough cake, starch paste, rice cakes, bean-jamfilled buns, sweet rice jelly, bean jelly, sweet bean paste, soft sweetbean paste, sweet balls, jelly, castella sponge cake and toffee;confectioneries such as bread, biscuits, crackers, cookies, pies,pudding, cream puffs, waffles, sponge cake, donuts, chocolate, chewinggum, caramel and candy; frozen desserts such as ice cream and sherbet;syrups such as fruit syrup and frozen nectar; spreads and pastes such asbutter cream, custard cream, flower paste, peanut paste and fruit paste;processed fruit or vegetable products such as jams, marmalades, syrupsand sweetened fruits; processed grain products such as bread, noodles,rice products and artificial meat products; fat and oil products such assalad oils and margarine; pickles such as pickled sliced vegetables,fresh radish pickles, pickled turnips and pickled scallions; picklestock such as pickled radish stock and pickled cabbage stock; livestockproducts such as ham and sausage; fish meat products such as fish ham,fish sausage, boiled fish paste, pounded fish cake and sea urchin eggpaste; delicacies such as salted cuttlefish or squid gut, vinegar-soakedtangle, dried cuttlefish strips and sundried blowfish; soy-boiledvegetables or fish made from laver, edible wild plants, driedcuttlefish, small fish or shellfish; vegetable foods such as boiledbeans, potato salad, tangle rolls and tempura; dairy products such ascooked eggs, milk beverages, butter and cheese; bottled or canned fishmeat, livestock meat, fruits and vegetables; liquor such as syntheticsake, flavored sake, fruit wine and western alcohol beverages; softdrinks such as coffee, cocoa, juice, carbonated beverages, lactic acidbeverages and lactic acid bacteria beverages; and various instantproducts such as pudding mixes, hotcake mixes, juice mixes, coffeemixes, red-bean soup mixes, soup mixes and the like. It may also beconveniently used as a vitamin C-fortifier, flavor enhancer,antioxidant, taste improver or the like in feed or fodder for breedinganimals such as livestock, poultry, bees, silkworms, fish and the like.

EFFECT OF THE INVENTION

The present invention provides the novel substance2-O-(β-D-glucopyranosyl)ascorbic acid, which has physiological actionsuperior to that of 2-O-(α-D-glucopyranosyl)ascorbic acid and isexpected to have applications in the fields of cosmetics, quasi drugs,medicines and foods, as well as2-O-(tetra-O-acetyl-β-D-glucopyranosyl)ascorbic acid as a novelintermediate thereof, a process for production of2-O-(β-D-glucopyranosyl)ascorbic acid using the intermediate and aprocess for production of 2-O-(β-D-glucopyranosyl)ascorbicacid-comprising compositions by extraction and purification from a plantof Lycium genus, and compositions comprising2-O-(β-D-glucopyranosyl)ascorbic acid derived from a plant of Lyciumgenus.

The invention further provides compositions comprising2-O-(β-D-glucopyranosyl)ascorbic acid or6-O-(β-D-glucopyranosyl)ascorbic acid obtained by glycosyltransferasereaction. It still further provides a method for easy removal ofcontaminants from solutions containing 2-O-(β-D-glucopyranosyl)ascorbicacid and industrial mass production of products with higher contents andhigher purity of 2-O-(β-D-glucopyranosyl)ascorbic acid.

EXAMPLES

The present invention will now be explained in greater detail throughexamples, with the implicit understanding that the scope of theinvention is in no way limited by these examples.

Example 1 Synthesis of2-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)ascorbic acid

After dissolving 5,6-O-isopropylideneascorbic acid (2 g, 9.3 mmol) inDMSO (20 ml), potassium carbonate (1.3 g, 9.4 mmol) and benzyl bromide(1.1 ml, 9.3 mmol) were added and the mixture was stirred at 50° C. for4 hours. Water was added to the reaction solution which was thenacidified with 1N HCl, extracted with ethyl acetate, washed with waterand then with saturated saline, dried with anhydrous MgSO₄, concentratedunder reduced pressure and purified by silica gel chromatography(AcOEt/n-hexane=3:1) to obtain 1.1 g of3-O-benzyl-5,6-O-isopropylideneascorbic acid (39% yield).

A mixture of this benzyl derivative (0.6 g, 2.0 mmol) and2,3,4,6-tetra-O-acetyl-1-O-(2,2,2-trichloroethoxycarbonyl)-β-D-glucopyranose(2.1 g, 4.0 mmol) was heated at 120-130° C. to melting. After 3 hours ofreaction, the reaction solution was purified by column chromatography(gradient from 25%-50% AcOEt/n-hexane) to obtain 850 mg of2-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-3-O-benzyl-5,6-O-isopropylideneascorbicacid (67% yield).

This glucoside (850 mg, 1.3 mmol) was dissolved in ethyl acetate (40ml), and 10% Pd—C (200 mg) was added for hydrogenolysis. After 2 hours,the catalyst was filtered out and the filtrate was concentrated to yieldapproximately 750 mg of2-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-5,6-O-isopropylideneascorbicacid.

The debenzylated compound (500 mg, 0.9 mmol) was dissolved in aceticacid (5 ml), water (5 ml) was added, and the mixture was heated at50-60° C. for 1.5 hours while stirring. After concentrating the reactionsolution, the obtained residue was extracted with ethyl acetate, washedwith water and then with saturated saline, dried with anhydrous MgSO₄and concentrated under reduced pressure, and then the obtained residuewas recrystallized from ethyl acetate/hexane to obtain 320 mg of2-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)ascorbic acid (48%yield).

¹H-NMR (δppm, DMSO-d₆); 1.94-2.01(12H), 3.42(3H, m), 3.7-4.3(4H, m),4.7-5.1(4H, m), 5.3-5.4(2H, m), 12.00(1H, br).

FABMS(+) m/z: 507.

Example 2 Synthesis of 2-O-(β-D-glucopyranosyl)ascorbic acid

After dissolving 2-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)ascorbicacid (300 mg, 0.6 mmol) in methanol (10 ml), a solution of potassiumcarbonate (600 mg) in water (9 ml) was added and the mixture was stirredfor 30 minutes. The reaction solution was neutralized with IR-120(H⁺),the resin was filtered off, and washing was performed with methanol anda 50% methanol aqueous solution. The filtrate and washing solution werecombined and concentrated, and then water was added and the mixture waslyophilized to obtain 2-O-(β-D-glucopyranosyl)ascorbic acid as amorphouscrystals (190 mg; 100% yield).

¹H-NMR (δppm, D₂O); 3.1-3.3(4H, m), 3.4-3.5(3H, m), 3.58(1H, d), 3.80(1H, t), 4.61(1H, d), 4.66(1H, d).

FABMS(−) m/z: 337.

Example 3 Measurement of 2-O-(β-D-glucopyranosyl)ascorbic acid Contentof a Plant of Lycium genus

Extracts obtained by immersing 3 g of different dry plants in a 10-foldvolume of 70% ethanol at room temperature for 7 days were diluted10-fold with 1.5% metaphosphoric acid/5 M KOH (pH 3.5) and used as testsamples for identification of naturally occurring2-O-(β-D-glucopyranosyl)ascorbic acid, based on the retention time of2.63 minutes in high performance liquid chromatography of chemicallysynthesized 2-O-(β-D-glucopyranosyl)ascorbic acid (LC-10Ai System byShimadzu Co., Ltd.; column: Inertsil ODS-3 (GL Science Co., Ltd.,4.6×150 mm, 5 μm), mobile phase: 20% MeOH-20 mM phosphate-5 mMtetra-n-amylammonium bromide, flow rate: 1.0 mL/min, column temperature:35° C., detection wavelength: 254 nm). As a result, a peak correspondingto 2-O-(β-D-glucopyranosyl)ascorbic acid was found in extracts of lyciifructus from Neimonggol Lycium barbarum L., Ningxia Lycium barbarum L.and Hebei Lycium Chinense Mill. The same was found in a sample similarlyprepared by adding a 2-fold amount of 70% ethanol to 100 g of freshfruit of Ningxia Lycium barbarum L. Each solid extract was measured bylyophilization and weight measurement after concentration of 5 mL of theliquid extract under reduced pressure. Taking into account thecalibration curves obtained using the solid extracts and chemicallysynthesized product, the concentrations of the extracts and the degreeof dilution, the contents of the extracts were determined to be from0.86% to 1.2%.

Example 4 Purification of 2-O-(β-D-glucopyranosyl)ascorbic acid in lyciifructus

After pulverizing 100 g of Ningxia Lycium barbarum L. with a ModelTS-10M tablet pulverizer by Tosho Co., Ltd., 800 mL of 30% ethanol wasadded for immersion at room temperature for 6 days, followed byfiltration, concentration under reduced pressure and lyophilization toobtain 65.7 g of product. A 1.94 g portion of the extract(2-O-(β-D-glucopyranosyl)ascorbic acid content: 0.86%) was dissolved indistilled water to make 40 mL (pH 4.5,; electric conductivity: 1.7mS/cm). The sample was passed through a Dowex 1-X8 column (acetate form,1.5×12 cm) at SV=1. It was then washed with an approximately 10 columnvolume (200 mL) of distilled water, subjected to linear gradient elution(100 mL×2) with 0-0.1 M acetic acid, subjected to linear gradientelution (100 mL×2) with 0.1-1.0 M acetic acid, and then eluted with 1.0M acetic acid. The absorbance at 280 nm was measured and the elution ofthe 2-O-(β-D-glucopyranosyl)ascorbic acid was examined by highperformance liquid chromatography in comparison with the retention timeof the chemically synthesized product. The apparatus and columntemperature were the same as in Example 1, but the other conditions werechanged to the following. Column: Inertsil ODS-3 (GL Science Co., Ltd.,3.0×150 mm, 5 μm), flow rate: 0.3 mL/min, detection wavelength: 245 nm,mobile phase: 2% MeOH-0.2 M KH₂PO₄/H₃PO₄ (pH 3.0)-0.2 mM EDTA-0.5 mMdodecyltrimethylammonium chloride. The retention time of the chemicallysynthesized product 2-O-(β-D-glucopyranosyl)ascorbic acid under theseconditions was 6.5 minutes. As a result of examination by highperformance liquid chromatography, the substance adsorbed to the columnwas found to have eluted in fractions 19-25 with 0.1-1.0 M acetic acidlinear elution (26 mg, 78% total yield for fractions 19-25, 50% purity).The results are shown in FIG. 1.

A portion of the fractions 19-25 corresponding to2-O-(β-D-glucopyranosyl)ascorbic acid was supplied to high performanceliquid chromatography to obtain a high purity product. The conditionswere as follows. LC System by Gilson Co. (Type 305 Master Pump, Type 116UV detector), column: ODS-UG-5 (4.6×250 mm, 5 μm, product of NomuraChemical Co., Ltd.), mobile phase: 5% methanol/20 mM ammonium formate/5mM di-n-butylamine acetate, flow rate: 0.5 mL/min, detection wavelength:254 nm, fractionation at 0.5 min increments using an FC-203 Type BFraction Collector (Gilson Co.). The corresponding fraction wasconcentrated under reduced pressure and lyophilized, dissolved inDeuterium Oxide and its nuclear magnetic resonance spectrum measured andcompared with the synthesized product. The results are shown in FIG. 2.

Example 5 Enzyme Synthesis of 2-O-(β-D-glucopyranosyl)ascorbic acid

Commercially available cellulase, β-glucosidase and β-glucanase enzymeagents were examined based on the retention time of 5.2 minutes forchemically synthesized 2-O-(β-D-glucopyranosyl)ascorbic acid in a LCSystem by Gilson Co. (Type 305 Master Pump, Type 116 UV detector),column: Inertsil ODS-3 (DL Science Co., Ltd., 4.6×150 mm, 5 μm), mobilephase: 20% MeOH-20 mM phosphate-5 mM tetra-n-amylammonium bromide, flowrate: 0.5 ml/min, detection wavelength: 254 nm). The enzyme reactionsystem was dissolved with 10 mM acetate buffer (pH 5.0) to 1 ml, for 0.3M cellobiose and 0.2 M ascorbic acid. A 50 μl portion of the enzymesolution was then added thereto and reaction was initiated at 37° C.After heating at 100° C. for 5 minutes to terminate the reaction, theproduced β-D-glucopyranosylascorbic acid was analyzed by highperformance liquid chromatography. As a result, β-transglucosylationactivity was found in the cellulase (Sigma), β-glucosidase (Toyobo,Nacalai Tesque), Cellulosin T2 (Hankyu Kyoei Bussan), cellulase“Onozuka” RS, “Onozuka” FA and Pancelase BR (Yakult Pharmaceutical Ind.Co., Ltd.). Free ascorbic acid appeared at the position of 4.0 minutes,but peaks were also observed at adjacent positions of 3.6 minutes and5.2 minutes, and these were designated as substance X and substance Y,respectively. The transglucosylation ratio for substance X was 15.7% andthat for substance Y was 0.8%. When substances X and Y were subjected toco-chromatography with chemically synthesized product, substance Xmatched the holding time for 6-O-(β-D-glucopyranosyl)ascorbic acid andsubstance Y for 2-O-(β-D-glucopyranosyl)ascorbic acid.

After removing the contaminating protein by using a UF membrane withcutting molecular weight of 10,000, the free ascorbic acid was removedby fractionation using high performance liquid chromatography [LC Systemby Gilson Co. (Type 305 Master Pump, Type 116 UV detector), column:SUGAR SH1011 (Showa Denko Co., Ltd.), mobile phase: 0.1 M acetic acid,flow rate: 0.5 mL/min, column temperature: 30° C., detection:differential refractometer, 0.25 ml fractionation]. The fractionscontaining substance X and substance Y eluted at 29-31, and a 24.7 pgsample was obtained at a 96% yield.

This was then supplied to high performance liquid chromatography toobtain a high purity product. The conditions were as follows. LC Systemby Gilson Co. (Type 305 Master Pump, Type 116 UV detector), column:ODS-UG-5 (4.6×250 mm, 5 μm, product of Nomura Chemical Co., Ltd.),mobile phase: 5% methanol/20 mM ammonium formate/5 mM di-n-butylamineacetate, flow rate: 0.5 mL/min, detection wavelength: 254 nm,fractionation at 0.5 min increments using an FC-203 Type B FractionCollector (Gilson Co.). The fractions corresponding to substance X andsubstance Y were concentrated under reduced pressure, lyophilized anddissolved in Deuterium Oxide, and the nuclear magnetic resonancespectrum was measured and compared with chemically synthesized2-O-(β-D-glucopyranosyl) ascorbic acid. In the HSQC spectrum, thechemical shifts of the 4-position, 5-position and 6-position carbons ofthe ascorbic acid moiety of the chemically synthesized product were 73,73 and 66 ppm, respectively, while for substance X the chemical shiftsof the 4-position, 5-position and 6-position were all 73 ppm and thus ashift towards lower magnetic field was seen only for the 6-positioncarbon. Substance X was therefore concluded to be6-O-(β-D-glucopyranosyl)ascorbic acid.

Substance Y matched in the comparison with one dimentional NMR spectrumof chemically synthesized 2-O-(β-D-glucopyranosyl)ascorbic acid, and wastherefore concluded to be 2-O-(β-D-glucopyranosyl)ascorbic acid. Theresults are shown in FIG. 3 and FIG. 4.

Example 6 Transferase Reaction Conditions

1) Ammonium Sulfate Fractionation

Cellulase agent (Sigma) was dissolved in 20 mM acetate buffer (pH 5.0)to a concentration of 4%, and ammonium sulfate solutions at 20% gradedsaturations were successively added to prepare 0-20%, 20-40%, 40-60% and60-80% saturated ammonium sulfate precipitation fractions. Afterdissolving each of the fractions in 20 mM acetate buffer (pH 5.0), thetransfer product was confirmed according to Example 5. As a result,transfer activity was found in the ammonium sulfate 20-40% saturatedfraction.

2) Effect of pH

The test substance was dissolved to 1 ml with 0.1 M acetate bufferadjusted to various pH levels, for concentrations of 0.3 M cellobioseand 0.2 M free ascorbic acid. A 50 μl portion of enzyme solution wasadded thereto for reaction at 37° C. for 40 hours. After the reaction,the (β-D-glucopyranosyl)ascorbic acid produced was analyzed by highperformance liquid chromatography.

The results are shown in Table 1. The transglucosylation products werefound at pH 3 or at a higher pH, and 2-O-(β-D-glucopyranosyl)ascorbicacid was produced at 0.8% in reaction at pH 5 and at 1.0% in reaction atpH 6. Also, 6-O-(β-D-glucopyranosyl)ascorbic acid was produced at 11.8%in reaction at pH 5 and at 11.2% in reaction at pH 6.

TABLE 1 pH AA (%) AA6βG (%) AA2βG (%) 2 99.3 0.7 0 3 93.5 6.2 0.3 4 87.911.6 0.5 5 87.4 11.8 0.8 6 87.8 11.2 1.0

3) Reaction with Ascorbic Acid Derivatives

The test substance was dissolved to 1 ml with 0.1 M acetate bufferadjusted to various pH levels, for concentrations of 0.3 M cellobioseand 0.2 M each of free ascorbic acid, sodium ascorbate, calciumascorbate, free isoascorbic acid and sodium isoascorbate. A 50 μlportion of enzyme solution was added thereto for reaction at 37° C. for20 hours, after which the (β-D-glucopyranosyl)ascorbic acid was analyzedin the same manner as described above. The results are shown in Table 2.Each ascorbic acid derivative acted as a transfer reaction acceptor,with each producing a 2-O-(β-D-glucopyranosyl) derivative. Table 2

TABLE 2 AA (%) AA6βG (%) AA2βG (%) Free ascorbic acid 91.2 7.9 0.9Sodium ascorbate 97.6 1.6 0.8 Calcium ascorbate 95.9 2.8 1.3 Freeisoascorbic acid 95.4 3.8 0.8 Sodium isoascorbate 98.4 0.8 0.8

4) Partial Purification

Cellulase agent (Sigma), Cellulase “Onozuka” RS and Pancelase BR (YakultPharmaceutical Ind. Co., Ltd.) were each applied to Q-Sepharoseion-exchange resin (Amersham Pharmacia Biotech Co.) equilibrated with 20mM acetate buffer (pH 5.0) after ammonium sulfate precipitation of theenzyme protein, and all of the transfer activity was found in theflow-through fractions. The results are shown in Table 3.

TABLE 3 AA (%) AA6βG (%) AA2βG (%) Cellulase (Sigma) 89.9 9.0 1.1Onozuka RS (Yakult) 86.4 11.6 2.0 Pancelase BR (Yakult) 94.0 5.4 0.6

5) Enzyme Immobilization

The enzyme agent Pancelase BR sold as a food additive contains, inaddition to the enzyme (5%), lactose at 95%. After adding 6 g ofPancelase BR enzyme agent to 60 ml of 20 mM acetate buffer (pH 5.0), themixture was applied to Marathon WBA (2 ml resin, product of Dow ChemicalCo.) equilibrated with the same buffer, and the flow-through fractionwas obtained. Ammonium sulfate was then added to 20% saturation, and themixture was immobilized on Chitopearl BCW3510 (2 ml resin, product ofFuji Spinning Co., Ltd.) equilibrated with 20% saturated ammoniumsulfate/20 mM acetate buffer (pH 5.0), to prepare the immobilizedenzyme. The immobilized enzyme resin was added to 10 ml of 20% saturatedammonium sulfate/20 mM acetate buffer (pH 5.0) dissolving 0.35 g ofascorbic acid and 1 g of cellobiose, and reacted at 37° C. The resultsare shown in Table 4.

TABLE 4 Reaction time AA (%) AA6βG (%) AA2βG (%) Day 1 94.1 4.8 1.1 Day2 91.2 7.6 1.2

Example 7 Purification of (β-D-glucopyranosyl)ascorbic acid

A 20 mg portion of Cellulase agent (Sigma) was dissolved in 1 ml of 20mM acetate buffer (pH 5.0), the mixture was applied to Marathon WBA (0.5ml resin, product of Dow Chemical Co.) equilibrated with the samebuffer, and the flow-through fraction was obtained. The enzyme solutionwas added to 10 ml of 20 mM acetate buffer (pH 5.0) which had dissolved0.35 g of ascorbic acid and 1 g of cellobiose, and the mixture wasreacted at 37° C. for 2 days to obtain a reaction solution containing11.8% 6-O-(β-D-glucopyranosyl)ascorbic acid and 0.8%2-O-(β-D-glucopyranosyl)ascorbic acid. The solution was filtered with aUF membrane to collect and remove the enzyme, and the resulting solution(pH 4.3, electric conductivity: 1.6 mS/cm) was passed through a Dowex1-X8 column (acetate form, 1.5×12 cm) at SV=2.5. It was then washed withan approximately 10 column volume (200 mL) of distilled water andsubjected to linear gradient elution (80 mL×2) with 0-0.1 M acetic acidand to linear gradient elution (80 mL×2) with 0.1-1.0 M acetic acid. Thefractionation successively produced high6-O-(β-D-glucopyranosyl)ascorbic acid content fractions (fractions65-68), a high unreacted ascorbic acid content fraction and high2-O-(β-D-glucopyranosyl)ascorbic acid content fractions (fractions101-108). Fractions 101-108 were collected as the high2-O-(β-D-glucopyranosyl)ascorbic acid content fractions (2.4 mg, 45%yield).

Example 8 Protecting Effect of 2-O-(β-D-glucopyranosyl) ascorbic acidAgainst Cell Death of Human Skin Epidermal Keratinocytes (HaCaT) Inducedby Ultraviolet B (UVB) Irradiation

The human skin epidermal keratinocyte line HaCaT (a cell line providedby Professor Fusenig of Heidelberg University) was plated on a 24-wellplate at 10,000 cells/well with Dulbecco's modified Eagle medium (DMEM)containing 10% fetal bovine serum (FBS) and after 18 hours, the cellswere irradiated with UVB (maximum wavelength: 312 nm) at 35millijoules/square centimeter (mJ/cm²). Two hours before irradiation,2-O-(β-D-glucopyranosyl)ascorbic acid was added at 20-100 μM and removedwith rinsing just before irradiation. The irradiation was performed inPBS in the absence of chemical agents, after which culturing wascontinued in DMEM containing 10% FBS, and the viable cell count wasdetermined 24 hours after irradiation by measuring the mitochondrialdehydrogenase activity using2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliummonosodium salt (WST-1). The results are shown in FIG. 5. Forcomparison, 2-O-(α-D-glucopyranosyl)ascorbic acid and ascorbic acid wereexamined in a similar manner, and the results are also shown in FIG. 5.

Example 9 Effect of 2-O-(β-D-glucopyranosyl)ascorbic acid onIntracellular Ascorbic Acid Concentrations in Human Skin EpidermalKeratinocytes

Human skin epidermal keratinocyte cells HaCaT were plated onto a 100 mmdiameter dish at a cell count of 370,000. After 16 hours of culturing,there was added 100 μM of 2-O-(β-D-glucopyranosyl)ascorbic aciddissolved in DMEM containing 10% FBS and 40% of 24-hour serum-free HaCaTculture solution. The medium was removed 3-24 hours after the addition,rinsing was performed twice with ice-cooled PBS, and the cell sheet wastreated with trypsin to obtain the separated cells. These were suspendedin PBS containing 50 μM dithiothreitol (DTT) and rinsed 3 times bycentrifugation. The cells received freeze-thawing twice were crushedwith a Potter-type teflon homogenizer for 30 sec on the ice. The cellhomogenate was centrifuged at 5° C., and supernate thus separated wasstored on the ice. The supernatant was subjected to treatment withMolcut (product of Nihon Millipore Co., Ltd.; High-pressureultrafiltration unit, nominal molecular weight limit (NMWL): 10,000,polyethersulfone membrane), and the amount of intracellular ascorbicacid was analyzed by high performance liquid chromatography (AS-8020System by Toso Co., Ltd., column: Shodex ODSpak (4.6×150 mm, product ofShowa Denko Co., Ltd.), mobile phase: 0.1 M KH₂PO₄—H₃PO₄ (pH 2.35)-0.1mM EDTA-2 Na, flow rate: 1.5 mL/min) using a coulometric electrochemicaldetector (ESA Co., Bedford, Mass., 200 mV). The results are shown inFIG. 6. For comparison, 2-O-(α-D-glucopyranosyl)ascorbic acid andascorbic acid were examined in a similar manner, and the results arealso shown in FIG. 6.

Example 10 Promoting Effect of 2-O-(β-D-glucopyranosyl)ascorbic acid onCollagen Synthesis by Normal Human Dermal Fibroblasts (NHDF)

Normal human dermal fibroblast cells (NHDF) were plated onto a 100 mmdiameter dish at a cell count of 370,000. After 16 hours of culture,there was added 100 μM of 2-O-(β-D-glucopyranosyl)ascorbic aciddissolved in DMEM medium containing 10% FBS and 40% of 24-hourserum-free NHDF culture solution. After another hour, 0.12 mL (120 μCi)of L-[2,3-³H]proline was added and culturing was continued for 48 hours.The medium was removed after culturing, and the cell sheet was rinsed 4times with PBS. The cells were then treated with trypsin, lysed with analkali, and then neutralized to obtain the intracellular protein. Thefraction obtained by digesting this protein with Clostridium collagenasewas measured in terms of radioactivity with a liquid scintillationcounter using Scintisol EX-H. The intracellular protein fraction nottreated with collagenase was also measured in terms of radioactivityusing a liquid scintillation counter. The difference in radioactivitieswas calculated and recorded as the collagen synthesizing activity. Theresults are shown in FIG. 7. For comparison,2-O-(α-D-glucopyranosyl)ascorbic acid and ascorbic acid were examined ina similar manner, and the results are also shown in FIG. 7.

Example 11 Intestinal Absorpition of 2-O-(β-D-glucopyranosyl)ascorbicacid in Rat

A solution of 2-O-(β-D-glucopyranosyl)ascorbic acid in Milli-Q UltrapureWater (Millipore Corporation) (100 mg/4 mL) was orally administered at adose of 100 mg/kg by using a feeding tube to overnight-starved and awake10-week-old male Wistar rats (n=3) purchased from Nihon Charles RiverCo. After 0, 0.5, 2 and 4 hours, heparinized blood samples werecollected from the portal vein and the plasma from each sample wasseparated by centrifugation (6000×g, 10 min). After addition of anequivalent of ice-cooled 10% metaphosphoric acid (containing 40 mMdeferoxamine mesylate), the mixture was centrifuged (10,000×g, 10 min)to obtain protein-removed portal vein plasma, of which the unconverted2-O-(β-D-glucopyranosyl)ascorbic acid and ascorbic acid concentrationswere measured by high performance liquid chromatography (LC-10Ai Systemby Shimadzu Co., Ltd., Column: Inertsil ODS-3 (GL Science Co., Ltd.,3.0×150 mm, 5 μm), mobile phase: 15% MeOH-17 mM KH₂PO₄/H₃PO₄ (pH 3.5)-5mM tetra-n-amylammonium bromide, flow rate: 0.3 mL/min, columntemperature: 35° C., detection wavelength: 254 nm). The unconverted2-O-(β-D-glucopyranosyl)ascorbic acid and the metabolite (ascorbic acid)were found present in maximum amounts 30 minutes after administration.The results are shown in FIG. 8.

These results demonstrated that the 2-O-(β-D-glucopyranosyl)ascorbicacid of the invention is a provitamin C with superior physiologicaleffects from the standpoint of stability and prolonged activity,compared to 2-O-(α-D-glucopyranosyl)ascorbic acid. It is thereforeexpected to have applications in the fields of foods, cosmetics, andquasi drugs or drugs. In addition,2-O-(tetra-O-acetyl-β-D-glucopyranosyl)ascorbic acid is a novelintermediate useful for production of 2-O-(β-D-glucopyranosyl)ascorbicacid. Compositions comprising 2-O-(β-D-glucopyranosyl)ascorbic acid canbe obtained by extraction from a plant of Lycium genus. In addition tochemical synthesis, 2-O-(β-D-glucopyranosyl)ascorbic acid can also beobtained by glycosyltransferase reaction.

Example 12 Effect of 2-O-(β-D-glucopyranosyl)ascorbic acid on PopulationDoubling (PDL) Level of Human Dermal Fibroblasts

Normal human dermal fibroblast (NHDF) cells were plated onto a 100 mmdiameter dish at a cell density of 370,000. After 16 hours of culture inDMEM supplied with 10% FBS, the medium was replaced by DMEM fortifiedwith 10% FBS and supplied with 40% of a domestic-conditioned mediumcontaining 100 μM of 2-O-(β-D-glucopyranosyl)ascorbic acid. Thecultivation was continued for 46-188 hours until cells reached a nearconfluent state. The domestic-conditioned medium was prepared byseparately culturing NHDF cells to the state of near confluency, whichwas further cultivated in a serum-free medium for 24 hours, andcollecting the culture supernatant, which was stored in a refrigeratorand used within 3 days. In connection with the above 46-188 hours ofcultivation, the medium was changed every 3 days with fresh DMEMfortified with 10% FBS and supplied with 40% of the domestic-conditionedmedium containing 100 μM of 2-O-(β-D-glucopyranosyl)ascorbic acid. Whenmedium change was made, the cell number in the old culture medium wascounted and used in the PDL assessment. The number of cells adheringonto the plate was counted by Coulter counter. PDL value at the start ofculture was taken as 0, and PDL values thereafter were calculated by theequation:PDL=log ₂(recovered cell number/seeded cell number).The results are shown in FIG. 9. For comparison, similar tests wereconducted substituting 2-O-(α-D-glucopyranosyl)ascorbic acid andascorbic acid for 2-O-(β-D-glucopyranosyl)ascorbic acid, and the resultsare also shown in FIG. 9.

Example 13 Effect of 2-O-(β-D-glucopyranosyl)ascorbic acid on theReduction Speed of Telomere Lengths

Genomic DNA extract from NHDF cells was treated with restriction enzymeswhich do not cleave telomere sequence, and a DNA fragment containing theentire length of telomere (Terminal Restriction Fragments, TRFs) wasprepared. The TRFs were separated by electrophoresis and the length ofthe telomere DNA was quantified with a labeled probe specificallybinding to telomere. Details of the method used in the study isdescribed in Life Sciences, Vol. 63, No. 11, 935-948 (1998).

Preparation of TRF from NHDF Cells and Agarose Gel Electrophoresis

The fibroblast cells (NHDF) from human skin epidermis were dispersed bytreatment with trypsin, added into a 1.5 ml tube at 10⁶/tube,centrifuged for 2 min. at 1,200 rpm at 4° C., and the supernatant wasdiscarded. The pellet was washed two times with 1 ml RNase free PBS(−),the supernatant was removed as sufficiently as possible, and the cellswere stored at −80° C. The frozen sample was restored to ambienttemperature, genomic DNA was extracted by use of IsoQuick Nucleic AcidExtraction Kit (ORCA Research Inc.). The extracted DNA was dissolved in10 mM Tris-HCl, 1 mM EDTA, pH8.0 to be stored at 4° C. The DNA contentwas determined by the fluorescent reader (Cytofluor 2350, MilliporeCorporation) with the DNA binding reagent Hoechist 33258, and TRF wasprepared by restricted digestion with HinfI treatment. The restricteddigestion was carried out as follows. There were added into a 1.5 mltube, 2 μl of 10× H buffer (TaKaRa, Kyoto), the solution of extractedgenomic DNA (2 μg), sterilized water to make up 19 μl, and finally 1 μlof HinfI (6 U/μl, TaKaRa, Kyoto). The reaction was carried out for 3-4hours at 37° C., and the mixture was stored at −20° C. An gel plate ofagarose (type I, Sigma) was prepared so that the agarose concentrationat the bridge and the bed would be 1% and 0.8%, respectively. 1× Boyerbuffer (50 mM Tris-HCl, 20 mM Sodium Acetate, 2 mM EDTA, 18 mM NaCl, pH8.0) was used. 0.5 μg/lane of 1 Kb DNA Ladder (GIBCO BRL) as a sizemarker and the sample in admixture with 3 μl of loading buffer wereapplied onto the gel, and electrophoresis was carried out for 20 hoursat 35 V/cm.

Transblot

Subsequent to the electrophoresis, agarose gel was cut off and stainedwith ethidium bromide (2 μg/ml) for 15 min., a photograph of the gel wastaken on the UV transilluminator. Then the gel was soaked in thedenaturation solution (0.2 N NaOH, 0.6 M NaCl) and was shaken at anambient temperature for 25 min., then it was rinsed with distilled waterfor once. The gel was soaked again in the neutralizing solution andshaken for 30 min. The gel was placed on a 3 MM filter paper which wasset in a blotting equipment filled with 6×SSC, while care being takennot to incorporate any air. Then a nitrocellulose membrane filter(OPTITRAN BA-S85, Schleicher & Schuel) pre-soaked with 6×SSC, a 3MMfilter paper pre-soaked with 6×SSC, a paper towel, a glass plate, and aweight (2 kg) was successively placed on the gel, and blotting wasperformed overnight. Subsequent to the blotting, the membrane filter wassoaked in 3×SSC, and the water was drained briefly, then the position ofthe well was checked on the UV transilluminator. The membrane filter wassandwiched between filter papers, and then it was heated at 80° C.overnight (baking).

Labeling the (TTAGGG), Probe and Hybridization

The baked filter was soaked in 3×SSC, then it was soaked in thehybridization buffer (10× Denhart solution, 1M NaCl, 50 mM Tris-HCl (pH7.4), 10 mM EDTA, 0.1% SDS, 50 μg/ml denatured salmon sperm DNA) andprehybridized by shaking at 65° C. for 3-4 hours. After theprehybridization, the membrane filter was put into a shield bag and then2 ml of the hybridization buffer to which [³²P] 5′-terminal labeled(TTAGGG)₄ probe (TaKaRa) and 1 μl of 10 mg/ml denatured salmon sperm DNAhas been added. The bag was closed in such a way that no bubbles wouldbe incorporated. Then the hybridization was performed by incubating thebag at 50° C. overnight.

Autoradiography

Following the hybridization, the membrane filter was soaked in a washingbuffer (4×SSC, 0.1% SDS) and shaken at 55° C. for 15 min. Afterrepeating the above process 4 times, the water on the filter was drainedsufficiently, and then the filter was set together with an X ray film(Scientific Imaging Film, Kodak) in the cassette with a intensifyingscreen, and autoradiography was performed overnight. The smeary appearedTRF density peak was detected with laser densitometer (Ultroscan XL,Pharmacia), and the mobility was determined.

The results are shown in FIG. 10. For comparison, the results of2-O-(α-D-glucopyranosyl)ascorbic acid, and ascorbic acid were obtainedand included in FIG. 10 as well.

1. A method of providing vitamin C to a subject in need thereofcomprising orally administering to the subject an effective amount of2-O-(β-D-glucopyranosyl) ascorbic acid represented by the followingformula (1):

or a biologically acceptable salt or ester thereof, or a compositioncomprising the same as a provitamin C.
 2. The method of claim 1, whereinthe composition is a vitamin C-fortified food.
 3. A method ofalleviating ultraviolet damage to the skin of a subject comprisingexternally administering to the skin of the subject the2-O-(β-D-glucopyranosyl)ascorbic acid of claim
 1. 4. A method ofwhitening the skin of a subject comprising administering to the skin ofthe subject the 2-O-(β-D-glucopyranosyl) ascorbic acid of claim
 1. 5. Amethod of treating skin wrinkles or sagging comprising externallyadministering to the skin of a subject an effective amount of2-O-(β-D-glucopyranosyl)ascorbic acid of claim
 1. 6. A method oftreating skin aging in a subject comprising externally administering tothe skin of the subject an effective amount of 2-O-(β-D-glucopyranosyl)ascorbic acid or a biologically acceptable salt or ester thereof ofclaim
 1. 7. A method of treating skin aging in a subject comprisingexternally administering to the skin of the subject a plant extractcontaining an effective amount of 2-O-(β-D-glucopyranosyl)ascorbic acid.8. A method of promoting collagen synthesis in a subject comprisingexternally administering to the skin of the subject an effective amountof 2-O-(β-D-glucopyranosyl) ascorbic acid or a biologically acceptablesalt or ester thereof.
 9. A method of prolonging a skin cell life spanin a subject comprising externally administering to the skin of thesubject an effective amount of 2-O-(β-D-glucopyranosyl) ascorbic acid ora biologically acceptable salt or ester thereof.
 10. A method ofsupporting life of a normal human dermal fibroblast cell in a subjectcomprising externally administering to the skin of the subject aneffective amount of 2-O-(β-D-glucopyranosyl) ascorbic acid or abiologically acceptable salt or ester thereof.
 11. A method offortifying a cosmetic, quasi drug, or external medicine, comprisingadding to the cosmetic, quasi drug, or external medicine, an effectiveamount of 2-O-(β-D-glucopyranosyl) ascorbic acid or a biologicallyacceptable salt or ester thereof.